IMPROVEMENT OF DIGESTIBILITY AND STRUCTURAL

CHANGES OF OIL PALM EMPTY FRUIT BUNCHES BY

Pleurotus floridanus AND PHOSPHORIC ACID PRETREATMENT

Dissertation Summary

As requirement for

Doctoral degree

Program Study of Bioteknology

Proposed by

Isroi

08/275457/SMU/00535

to

Graduate Schoof of

UNIVERSITAS GADJAH MADA

YOGYAKARTA

2013

IMPROVEMENT OF DIGESTIBILITY AND STRUCTURAL

CHANGES OF OIL PALM EMPTY FRUIT BUNCHES BY

Pleurotus floridanus AND PHOSPHORIC ACID PRETREATMENT

Abstract

Oil palm empty fruit bunches (OPEFB) is abundance and not optimally

utilized as lignocellulose-based products. OPEFB has low digestibility and difficult to

process into its derivatives. This study aims to improve digestibility of OPEFB

through biological pretreatment with white-rot fungi. Stages of this study were (1)

selection white-rot fungi which selectively degrade lignin, (2) enhancement the

digestibility of OPEFB by Mn2+

and Cu2+

addition, (3) improvement digestibility of

OPEFB by a combination of biological pretreatment with phosphoric acid

pretreatment. All the studies were conducted on laboratory scale. Observations were

made on the changes of dry weight, lignin, cellulose, hemicellulose, physical

structure by SEM analysis, functional groups by FTIR analysis, and the crystallinity

of cellulose. Screening is done on Polyota sp, Pleurotus sp and sp Agraily. Pleurotus

sp chosen for further experiments and identified as P. Floridanus LIPIMC966,

because it can alter the lignin content from 19.63% to 15.22%, hemicellulose from

14.77% to 12.63%, and increase cellulose from 39.92% to 56.04%. The addition

effect of Mn2+

and Cu2+

on the biological pretreatment using P. Floridanus could

reduces the dry weight from 27.43% to 32.88%; lignin content up to 43.17% (Mn2+

)

and 34.08% (Cu2+

), hemicellulose content up to 32.82%, while cellulose content

remained constant. Combination of biological pretreatment and phosphoric acid were

evaluated based on the changes in components of lignocellulose, a structural and

morphology. Carbohydrate degradation after biological pretreatment, phosphoric

acid, and a combination of biology and phosphoric acid are 7.88%, 35.65%, and

33.77%, respectivelly. Pretreatment changed the hydrogen bonding of the cellulose

and linked between lignin and carbohydrates, which is related to the crystallinity of

the cellulose. The crystallinity of the cellulose as indicated by lateral order index after

pretreatment are 2.77 (without pretreatment), 1.42 (biology), 0.67 (phosphoric acid),

and 0.60 (a combination of biology and phosphoric acid), respectively. Phosphoric

acid pretreatment damaged the structure and morphology of the OPEFB fibers shown

by SEM analysis. Pretreatments have increased digestibility of the OPEFB 4

(biology), 6.3 (phosphoric acid), and 7.4 (biology and phosphoric acid)-fold

compared with whitout pretreatment.

Keywords: oil palm empty fruit bunches, Pleurotus floridanus, Cu, Mn, structural

changes, digestibility

1

INTRODUCTION

Oil palm empty fruit bunches (OPEFB) are abundant and not optimally

utilized as lignocellulose-based product. Indonesia is the largest palm oil producer in

the world that produces 20.7 million metric tons of OPEFB (FAOSTAT

2012). OPEFB are composed of 39.13% cellulose, 23.40% hemicellulose, and

34.37% lignin (Isroi et al. 2013 ). Having high carbohydrate content makes OPEFB

are potential as a feedstock of lignocellulosic derived products. OPEFB has low

digestibility and difficult to be processed into its derivatives product. The low

lignocellulosic digestibility caused by several factors, such as: the content and

composition of lignin, cellulose crystallinity, degree of polymerization, pore volume,

acetyl groups bound to the hemicellulose, surface area and particle size of the

biomass (Alvira et al. 2010, Anderson and Akin 2008, Rivers and Emert

1988). OPEFB require pretreatment stage to change the structure and to break down

the lignin barrier making cellulose more accessible to hydrolytic enzymes. Research

to get the right pretreatment method for OPEFB is needs to be done, so that the high

potential OPEFB could utilize into lignocellulosic derivative products.

Pretreatment of lignocellulose can be done through physical, chemical and

biological methods or a combination of these methods (Alvira et al. 2010, Taherzadeh

and Karimi 2008). Biological pretreatment utilize the ability of white rot fungi

(WRF) or its enzymes to break down lignin and altered the structure of lignocellulose

(Hatakka A.I. 1983, Taniguchi et al. 2005). Biological pretreatment has several

advantages such as: i) low energy requirement, ii) low capital investment, iii) no

chemical requirement, iv) mild environmental conditions, v) specific to substrate, vi)

simple process and equipment requirement (Kirk & Chang, 1981; Sun & Cheng,

2002). WRP are grouped into selective and non selective groups. WRP selective is WRP that degrade lignin more than cellulose and hemicellulose, whereas non-

selective is WRP that degrade all components of lignocellulose on comparable

amount. Biological pretreatment influenced by several factors, such as the addition of

cations (Mn2+

dan Cu2+

) (Camarero et al. 1996, Palmieri et al. 2000). The addition of

cations could increase production and activity of ligninolytic enzyme, lignin

degradation and improve digestibility of cellulose. Some isolates WRP successfully

isolated by Indonesian Biotechnology Research Institute for Estate Crops ( IBRIEC )

and have the ability to degrade lignin, i.e.: Polyota sp , sp Agraily , and Pleurotus sp .

Selectivity of these WRP isolates is unknown. Selection of the appropriate selective

WRP isolates for OPEFB is needed to develop biological pretreatment method with

the addition of cations (Mn2+

dan Cu2+

).

Biological pretreatment has some disadvantages compared with the

physical/chemical pretreatment, such as: lower of sugar yield (Taherzadeh & Karimi,

2008). Digestibility of lignocellulose could be improved through a combination of

biological pretreatment with chemical pretreatment (Itoh et al. 2003, Ma et al. 2010,

Taniguchi et al. 2010, Yu et al. 2010). One of the chemicals that can be used for

2

pretreatment is phosphoric acid. Phosphoric acid can solubilize cellulose and

fractionate lignocellulose. Phosphoric acid pretreatment reported efficient in reducing

cellulose crystallinity and increase the production of biogas from OPEFB (Nieves et

al. 2011). Phosphoric acid pretreatment of lignocellulosic materials has been reported

to increase fractionation and digestibility of lignocellulose (Zhang et al. 2007).

Combination of biological pretreatment with phosphoric acid pretreatment needs to

be tested in order to improve OPEFB digestibility. Combination of biological

pretreatment with phosphoric acid pretreatment has been not reported in the literature.

Lignocellulosic biomass undergoes physical and chemical changes after

pretreatment. These changes include alteration in the content of lignin, cellulose,

hemicellulose, reduction of cellulose crystallinity, pore area increase, damage to the

surface area and also changes in the functional groups. Analysis of physical and

chemical changes in the structure and composition of OPEFB after pretreatment is

needed to understand the mechanism of increasing of OPEFB digestibility and

designing appropriate pretreatment to produce optimal pretreatment process.

General aims of this study is to increase digestibility of OPEFB with a

combination of biological pretreatment using WRP and phosphoric acid pretreatment.

WRP was selected from three collections of WRP isolates. Cation (Mn2+

dan Cu2+

)

was added to biological pretreatment in order improve delignification of OPEFB.

Alteration in lignin, cellulose, hemicellulose, the degree of crystallinity, changes in

the physical structure and functional groups were analyzed to determine the

characteristics changes that contribute to increasing the digestibility of OPEFB.

MATERIALS AND METHODS

1.1. Microorganisms and Medium

1.1.1. Microorganisms Pleurotus sp, Polyota sp and sp Agraily were obtained from the Indonesian

Biotechnology Research Institute for Estate Crops (IBRIEC). All WRPs were grown

and maintained using Potato Dextrose Agar media (PDA, Badco) and incubated for

approximately one week before being used as inoculum for biological pretreatment.

Pleurotus sp identified by LIPIMC (LIPI Microbial Collection) as Pleurotus

floridanus LIPIMC996.

Yeast Saccharomyces cerevisiae CBS 8066 was obtained from Centraalbureau

voor Schimmelcultures (Delft, the Netherlands). Yeast cultures maintained on YPD

agar medium containing 20 g/L agar (Scharlau), 10 g/L yeast extract (Scharlau), 20

g/L peptone (Fluka), and 20 g/L D-glucose (Scharlau) as a source carbon and stored

at 4 C.

1.1.2. Media Medum for the growth and maintenance of WRP was a medium potato

dextrose agar (PDA, DIFCO Laboratories, Detroit, MI). The composition of liquid

3

medium for biological pretreatment were: (a) medium 1: 7 g/L KH2PO4, 1.5 g/L

MgSO4.7H2O, 1.0 g/L CaCl2.H2O; (b) medium 2: 7 g/L KH2PO4, 1.5 g/L

MgSO4.7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L CuSO4.5H2O; (c) medium 3: 7 g/L

KH2PO4, 1.5 g/L MgSO4. 7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L MnSO4.H2O; (d)

medium 4: 7 g/L KH2PO4, 1.5 g/L MgSO4.7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L

CuSO4.5H2O, 0.015 g/L MnSO4.H2O. Pretreatment OPEFB in the control treatment

without microbial inoculation media was added 1. Medum 1, 2, and 3 was used in the

phase 1 and 2. Medium 4 was used in the Phase 3.

1.1.3. Tandan Kosong Kelapa Sawit (TKKS) OPEFB obtained from palm oil mill Doloksinumbah plantation, PTPN IV,

North Sumatra. OPEFB chopped approximately 5 cm and sun dried (moisture content

4

Figure 1. Flow chart and phases of the research

1.3. Pretreatment Methods

Biological pretreatment for research on Phase 1 and 2 were conducted on solid

state fermentation without aeration and without stirring. Fifty gr of OPEFB was

weighted and added to the liquid media OPEFB appropriate treatment to reach 60%

moisture content. OPEFB further sterilized using an autoclave at a temperature of

121oC for 30 minutes. Four pieces of culture WRP ( 5mm) were inoculated

aseptically. The cultures were incubated at room temperature for 6 weeks. OPEFB

was harvested, washed, dried, and milled at the end of the incubation and then used

for dry weight and other lignocellulosic components analysis.

Biological pretreatment for Phase 3 studies conducted with solid state

fermentation without aeration and without stirring. Selected WRP isolated that

obtained from phase 1 was used on OPEFB biological pretreatment. Two hundred gr

of OPEFB was added 120 mL media then sterilized. Water content in the media

OPEFB was approximately 60%. Culture of P. floridanus LIPIMC996 was inoculated

aseptically. The cultures were incubated at 31C for 28 days in an incubator cabinet.

OPEFBs were harvested at the end of the incubation and frozen at temperatures

5

1.4. Phosphoric Acid Pretreatment Methods

Phosphoric acid pretreatment wes carried out according to the method

described in reference (Nieves et al. 2011, Zhang et al. 2007). Samples were stored at

temperatures < 0 C before used for hydrolysis or further analysis.

1.5. Enzymatic Hydrolysis

Enzymatic hydrolysis OPEFB Sample on Phase 2. Enzymatic hydrolysis

OPEFB sample was based on the method of NREL (Renewable Energy Laboratory,

USA) with slight modifications (Selig et al. 2008). Hydrolytic enzymes used were a

commercial enzyme (Cellulast , 64 FPU / ml and 58pNPGU/ml - glucosidase ,

Novozyme Co. ) at a dose of 60 FPU enzyme / g cellulase and 64 pNPGU / g -

Glucosidase. All samples were shaken with a shaker water bath at 50oC for 72 hours

and then filtered. Supernatant obtained was then used for glucose analysis.

Digestibility ( % ) was calculated by the following equation:

(1)

where glucose ( g ) was the mass of glucose in the fluid after hydrolysis and

cellulose ( g ) was the mass of cellulose in the substrate.

Enzymatic hydrolysis samples OPEFB in Phase 3 studies using the same

method as above with a few modifications. Hydrolytic enzymes used were

commercial enzyme Cellic CTec2 (148 FPU / mL , Co Novozymes , Bagsvaerd ,

Denmark ) at a dose of enzyme 30 , 60 , and 90 FPU / g cellulose. Digestibility (%) of

the initial cellulose was calculated by dividing the glucose produced by the initial

cellulose that is used by the following equation:

(2)

where glucose (g) is the amount of glucose in the fluid after the initial

cellulose hydrolysis and ( g ) is the content of cellulose in OPEFB before getting

pretreatment. All experiments were carried out duplicate and error bar was presented

as the standard deviation.

1.6. Simultaneous Saccharification and Fermentation

Simultaneous Saccharification and Fermentation (SSF) carried out by the

method of NREL (Dowe and McMillan 2008) using a commercial enzyme Cellic

CTec2 ( 148 FPU / mL , Co Novozymes , Bagsvaerd , Denmark ) at a dose of 60 fpu

enzyme / g cellulose. Cellulose concentration used was 6% in the 0:05 M citrate

buffer pH 4.8. SSF done with volume 100ml to 250ml erlemeyer equipped with a gas

trap (bubble trap). SSF was carried out at 31oC in a water bath shaker for 72 hours.

Ethanol production was observed every day.

1.7. Analytical Methods

Chemical analysis of the OPEFB components (lignin, hemicellulose, and

6

cellulose) in Phase 1 and 2 studies were conducted using Chesson - Datta methods

(Datta 1981). Cellulose, hemicellulose, and lignin content of OPEFB on Phase 3

study was determined by the method of NREL (Sluiter A. et al. 2011). Ash was

determined using the furnace overnight at 575 C (Sluiter A. D. et al. 2008). Dry

weight was determined as oven dry weight (ODW) after drying the sample at 105

3C for 24 hours in accordance with TAPPI method T264 cm - 97 standard test

(TAPPI 2002).

Fungal growth during the pretreatment was estimated based on the dry weight

of fungal biomass (Kumar et al. 2006). Biomass analysis was conducted to study the

Phase 1 and Phase 2.

Changes in functional group observed by the adsorption change of the IR

spectrum (infra red) by OPEFB samples at a specific wavelength ( Jeihanipour ,

Karimi et al . 2009). IR spectra measurements was performed using FTIR

spectrometer ( Impact 410 , Nicolet Instrument Corp. , Madison , WI ) , 32 scans ,

resolution 4 cm - 1

in the range of 600-4000 cm - 1

and controlled by softwere Nicolet

OMNIC 4.1 ( Nicolet Instrument Corp. , Madison , WI ) and analyzed using eFTIR

( EssentialFTIR , USA ) softwere.

Evaluation of changes in the physical structure of the OPEFB sample surface

before and after pretreatment was visualized using Scanning Electron Microscopy

analysis (SEM) Model JEOL JSM - 820 ( JEOL Ltd. , Akishima , Japan).

Monosaccharides (cellobiose, glucose, xylose, mannose, Galactose and

Arabinose) were analyzed using HPLC system equipped with an autosampler

(WalterTM 717, Milford, USA), UV detector (WalterTM 485, Milford, USA) , and

ELS detector (WalterTM 2424, Milford, USA) . Monosugar separated using a Bio -

Rad Aminex HPX - 87P column (Aminex HPX - 87P, Bio - Rad, USA) , pure water

as the mobile phase with a flow rate of 0.6 ml min - 1 under isothermal conditions at

85oC . A Bio - Rad Carbo - P column protector (column guard, Bio - Rad, USA) was

used and placed outside the main column at room temperature.

Ethanol concentrations were analyzed using HPLC system equipped with an

autosampler ( WalterTM 717, Milford , USA), UV detector (WalterTM 48, Milford,

USA), and ELS detector (WalterTM 2424, Milford, USA). Monosugar separated

using a Bio - Rad Aminex HPX - 87H column ( Aminex HPX - 87H , Bio - Rad, USA

) , 0.025M H2SO4 as the mobile phase with a flow rate of 0.6 ml min - 1

under

isothermal conditions at 85oC . A Bio - Rad Carbo - P column protector (column

guard, Bio - Rad , USA ) was used and placed outside the main column at room

temperature . Ethanol standards were dissolved in 0.025M H2SO4 concentration in

some and used as a comparison to calculated ethanol concentration in the sample.

The data were analyzed by statistical analysis. Data shown were the average

of each test. Value of standard deviation (SD) were calculated and displayed to

determine the error of each data. Each data also performed analyzes of variance

(analysis of variance, ANOVA) to determine the significance different of treatment

effects on control. Correlation analysis of data from several experiments conducted to

determine the statistical relationships between data.

7

RESULTS AND DISCUSSIONS

1.8. White-rot fungi Selection for Biological Pretreatment of Oil Palm Empty

Fruit Bunch

OPEFBs were changes physically and chemically after biological

pretreatment. Pretreated OPEFB becomes brighter and softer than un-pretreated

OPEFB. Visual color change is one of the characteristics of lignocellulosic

degradation by WRP (Hatakka Annele 2001). Decreased levels of lignin by WRP

likely cause discoloration of the lignocellulose (Bajpai 2004, de Jong et al. 1997).

Changes in lignin, cellulose, and hemicellulose content are shown in Figure 2.

The content of lignin and hemicellulose decreased significantly, whereas cellulose

increased significantly after biological pretreatment. Lignin content decreased

significantly from 19.63% (initial content) up to 15.32% (Pleurotus sp), 16.63%

(Polyota sp) and 18.07% (Agraily sp). Hemicellulose content from the lowest on each

treatment were Pleurotus sp (12.63%), Polyota sp (14.26%) and Agraily sp (15.18%).

The content of cellulose (%) on each treatment were Pleurotus sp (56.04%), Agraily

sp (44.13%) and Polyota sp (42.03%).

Figure 2. The percentage of lignin, hemicellulose, and cellulose content of palm

empty fruit bunches (OPEFB): (a) without biologIcal pertreatment

(control), (b) Pleurotus sp , (c) Polyota sp , (d) Agraily sp. Biological

pretreatment was performed on solid state fermentation without aeration

and at room temperature for 4 weeks.

All third isolates of WRP could degrade lignin, but which showed the highest

decrease in lignin was Pleurotus sp. The content of hemicellulose (%) showed a slight

decrease in each isolate WRP. Hemicellulose degradation occurs in the same relative

proportion to the degradation of biomass, so the percentage of hemicellulose content

to the total biomass decreased slightly. Changes in cellulose content (%) after

pretreatment OPEFB was vary for each isolate WRP. Increasing in the percentage of

0

10

20

30

40

50

60

70

Kontrol Pleurotus sp Polyota sp Agraily sp

Kan

du

ng

an

(%

)

Lignin

Selulosa

Hemiselulosa

8

cellulose after pretreatment biological biomass also reported in reference (Xu et al.

2010). This increasing occurred due to degradation of other components (lignin and

hemicellulose) were higher than the degradation of cellulose, so it would be

proportionally increased the cellulose content. Pleurotus sp degraded lignin

approximately 22 % of the initial content of lignin and the results were comparable

with the results reported in the literature, namely by 25 % after biological

pretreatment for 60 days (Taniguchi et al. 2005).

Highest decreasing in lignin and hemicellulose content, and the highest

increasing in cellulose content by Pleurotus sp suggested that Pleurotus sp was more

selective in degrading lignin than the two other isolates. Similar results were also

reported by Kerem et al. (1992) that P. ostreatus selectively degrade lignin more than

Phanerochaete chrysosporium. Some literature reported that Pleurotus sp isolates

produced ligninolytic enzymes (Lac, MnP , and VP) as well as hydrolytic enzymes

(Chen et al. 2010, Goudopoulou et al. 2010, Martnez et al. 2005, Tinoco et al. 2011).

Pleurotus sp isolates were selected for further biological pretreatment OPEFB phase

2 & 3 and identified as Pleurotus floridanus with collection number LIPIMC 966.

1.9. Effect of Addition of Manganese (Mn2+

) and copper (Cu2+

) on Biological

Pretreatment of Oil Palm Empty Fruit Bunch Using Pleurotus floridanus

LIPIMC966

1.9.1. Effect of Biological Pretreatment on Dry Weight and Lignocellulosic Components

Dry weight of OPEFB after biological pretreatment for 42 days incubation

are shown in Figure 3. It is generally observed that all three treatments results in the

reduction of ODW of OPEFB, with the total reductions are 32.88%, 29.08%, and

27.43% for treatment with Mn2+

addition, treatment with Cu2+

addition, and treatment

with no nutrient addition, respectively. The ODW decline was a decreasing in the

total of lignocellulosic biomass includes reduced lignin content, cellulose,

hemicellulose, and other components. WRP degraded solid components into less

complex structures, water soluble materials and gaseous products that result in

decreasing of lignocellulosic biomass dry weight.

Pretreated OPEFB were analyzed for its components, i.e. hot water soluble

(HWS) materials, hemicelluloses, cellulose, and lignin. The data are presented in

Figure 4. It is shown that all components reduced subjects to biological degradation

by P. floridanus in all three different treatments at different rate of reduction. Hot

water soluble (HWS) consists of several components, such as carbohydrates, proteins,

and inorganic compounds. Activity of P. floridanus in this work has reduced the HWS

up to about 50% during 42 days of incubation. Both treatments with Mn2+

addition

and Cu2+

addition have accelerated the HWS reduction to some extends (Figure 4 A).

9

Figure 3. Decrease of dry weight (ODW) of oil palm empty fruit buches (OPEFB)

during pretreatment using Pleurotus sp with (a) without nutrient

addition (control), (b) CuSO4 (Cu2+

), and (c) MnSO4 (Mn2+

). Biological

pretreatment performed by solid state fermentation without aeration and

at room temperature

Similar results are also found for degradation of hemicellulose (Figure 4B)

and lignin (Figure 4D), in which the components are reduced and the addition of

Mn2+

increased the reduction rate. Mn2+

treatment showed a faster decline rate

compared to other treatments at day 21 and then remained relatively constant until

day 42. Control treatment showed a slower rate of decline, but the decline continued

until day 42 and a decrease in the biggest hemicellulose than other treatments.

However, as shown in Figure 3C, all three treatments shown slightly reduction

on the content of cellulose in the OPEFB (Figure 4 C). Decrease in hemicellulose and

lignin content on this phase 2 study confirm the phase 1 experiment showed that

isolates of P. floridanus was more degrade hemicellulose and lignin than cellulose. In

other words P. floridanus was more selective in lignin degradation, HWS, and

hemicellulose than cellulose.

The fact that addition of Mn2+

and Cu2+

accelerates the degradation of most

components in lignocellulosic materials by fungi has also been observed by other

researchers (Janusz et al. 2006, Levin et al. 2007, Tychanowicz et al. 2006). Addition

of certain concentration of Mn2+

and Cu2+

can induce and control ligninolytic

enzymes production resulted in improvement of lignin degradation. Mn2+

concentration can affect MnP and LiP activities, whereas Cu2+

can affect Lac

activities (Isroi et al. 2011). Mn in the growth medium plays an important role in

regulating manganese peroxidase (MnP) and lignin peroxidase (LiP) activities. MnP

production dominates in the presence of Mn2+

, and conversely LiP production

dominates in the absence of Mn2+

. MnP can diffuse into the lignified cell wall and

oxidises non phenolic structure compounds. Laccase oxidises phenolic structures of

lignin.

0

5

10

15

20

25

30

0 7 14 21 28 35 42 49

Be

rat

Ke

rin

g (

gr)

Hari ke-

Kontrol Cu Mn

10

Figure 4. Change component content OPEFB , ie : hot water soluble (HWS) (A) ,

hemicellulose (B) , cellulose (C) , and lignin (D) during pretreatment

with Pleurotus floridanus LIPIMC996 without the addition of cations

(control), with the addition of CuSO4 (Cu2+

) , and the addition of

MnSO4 (Mn22+

) . Biological pretreatment performed by solid

fermentation culture, without aeration , and at room temperature.

1.9.2. Effect of Biological Pretreatment on Physical and Structural Characteristics

Inoculation of OPEFB with P. floridanus LIPIMC996 results in changing on

physical characteristics of OPEFB, i.e. it turns into lighter color (from dark brown), it

is more brittle and easier to grind. The colour change may be used as an indication of

lignin reduction or removal.

The peak of IR Spectrum at certain wavelength could lower, higher and/or

shifted which indicates the alteration of certain functional groups associated with that

wavelength. Analysis of FTIR spectra shown in Figure 5 and bands assignment are

described in Table 1. Some bands associated with polysaccharides and cellulose were

little changed for all pretreatment, namely: 3450-3000, 1456, 1162-1158, 897, and

769 cm - 1

. These adsorption bands were consistent with the content of cellulose

OPEFB that were not degraded by fungi (Figure 4 C). Peak at 640 cm - 1

, 760 cm - 1

and 1,366 cm - 1

are associated with significant changes in cellulose structure after

pretreatment. Intensity at a wavelength of 1739-1738 cm - 1

(polysaccharide)

significantly decreased after pretreatment. Bonds between lignin and carbohydrates

may exist in this peak (Takahashi dan Koshijima 1988). Hemicellulose and lignin

degradation by fungi can break the bond between carbohydrates and lignin that can

11

contribute to the decrease in adsorption at 1739-1738 cm - 1

peak.

Figure 5. FTIR spectra of biological pretreated OPEFB with P. floridanus in the

control treatment, and Cu 2+

and Mn2+

for: a) 0 days, b) 7 days, c) 14

days d) 21 days, and e) 28 days.

Crystallinity of cellulose could be predicted using intensities ratio of certain

bands at the IR spectra, that are A1418/A895 known as Lateral Order Index (LOI)

(O'Connor, Dupre et al 1958;. Hurtubise dan Krassig 1960). LOI value of biological

pretreated OPEFB shown in Figure 9. Crystallinities of cellulose were decreased

during pretreatment. Meanwhile, decreasing rate of OPEFB pretreated with Mn2+

and

Cu2+

addition shown higher than without cations addition. As indicating by FTIR

analysis of cellulose IR band, although there is no significant degradation of cellulose

but structure of the cellulose could be changes, such as their crystallinity.

Bands at wavelengths of 1,595 and 1,505 cm-1

are associated with significant

changes of lignin after pretreatment with Mn2+

and Cu2+

. Meanwhile, the intensity at

1.032 cm-1

also decreased after pretreatment with the addition of Mn 2 Absorption of

IR spectra at wavenumber 1422-1424 cm-1

indicating presence of the syringyl type

lignin (the major type of hardwood lignin) that absorb only at near band 1230 cm-1

(Pandey and Pitman 2003). The observation at these wavenumber showed significant

decrease in the lignin content, indicating loss of C-C, C-O, and C=O stretching (G

condensed > G estherified). Through FTIR spectra analysis, biological pretreatment

12

of OPEFB displayed significant changes in its functional groups in various regions,

particularly in G unit and S unit of lignin, suggesting deformation of biomass during

biological pretreatment.

Table 1. Assignment of FTIR-Absorption Bands (cm-1

) to various components of

oil palm empty fruit bunches according to literature

Wavenumber

(cm-1

)

Assignments Source Ref.

670 C-O out-of-plane bending

mode

Cellulose (Schwanninger et

al. 2004)

715 Rocking vibration CH2 in

Cellulose I

Cellulose (Schwanninger et

al. 2004)

858-853 C-H out of plane deformation

in position 2,5,6

G-Lignin (Fackler et al. 2010)

897 Anomere C-groups C(1)-H

deformation, ring valence

vibration

Polisakarida (Fackler et al. 2010,

Fengel 1992)

996-985 C-O valence vibration (Schwanninger et

al. 2004)

1035-1030 Aromatic C-H in plane

deformation, G>S; plus C-O

deformation in primary

alcohols; plus C=O stretch

(unconj.)

Lignin (Schwanninger et

al. 2004)

1162-1125 C-O-C assimetric valence

vibration

Polisakarida (Fackler et al. 2010,

Schwanninger et al.

2004)

1230-1221 C-C plus C-O plus C=O

strech; G condensed > G

etherified

Polisakarida (Fackler et al. 2010,

Fengel 1992)

1227-1251 C=O stretch, OH i.p. bending (Faix O. and

Bttcher 1992)

1270-1260 G-ring plus C=O strectch G-Lignin (Faix O. 1991)

1315 O-H blending of alcohol

groups

Karbohidrat (Fackler et al. 2010)

1375 C-H deformation vibration Cellulose (Fengel 1992)

1470-1455 CH2 of pyran ring symmetric

scissoring ; OH plane

deformation vibration

(Schwanninger et

al. 2004)

1430-1416 Aromatic skeletal vibrations

with C-H in plane deformation

CH2 scissoring

Lignin (Faix Oskar et al.

1991)

1460 C-H in pyran ring symmetric

scissoring; OH plane

deformation vibration

Cellulose (Fengel 1992)

13

Wavenumber

(cm-1

)

Assignments Source Ref.

1515-1505 Aromatic skeletal vibrations;

G > S

Lignin (Faix Oskar et al.

1991)

1605-1593 Aromatic skeletal vibrations

plus C=O stretch; S>G; G

condensed > G etherified

Lignin (Faix Oskar et al.

1991)

1675-1655 C O stretch in conjugated p-

substituted aryl ketones

Lignin (Faix Oskar et al.

1991)

1738-1709 CO stretch unconjugated

(xylan)

Polisakarida (Faix Oskar et al.

1991)

2940-2850 Asymetric CH2 valence

vibration

(Schwanninger et

al. 2004)

2980-2835 CH2, CH2OH in Cellulose

from C6

Cellulose (Schwanninger et

al. 2004)

2981-2933 Symmetric CH2 valence

vibration

(Schwanninger et

al. 2004)

3338 Hydrogen bonded O-H

valence vibration;

O(3)H...O(3) intermolecular in

cellulose

Cellulose (Schwanninger et

al. 2004)

1.9.3. Effect of Biological Pretreatment on Digestibility OPEFB digestibility calculated by equation 1 was shown in Figure 6. OPEFB

digestibility increased with increasing incubation time of biological pretreatment

using P. floridanus. As shown in the figure, all samples show no significant difference

on its digestibility at 0 day of incubation, i.e. between 17.22 22.00 %. Sample pretreated with no cation addition could reach digestibility of 30.97% following 28

days of incubation. Sample treated with Cu and Mn addition have maximum

digestibility of about 60.27% and 55.67%, respectively. The fact that samples

pretreated with Cu2+

and Mn2+

addition have higher percentage of digestibility

indicates that Cu2+

and Mn2+

addition increased their susceptibility to hydrolysis. The

increase of digestibility is significantly observed for hydrolysis period up to 21 days,

after that the digestibility only changed slightly.

14

Figure 6. Hydrolysis results OPEFB samples that have received biological

pretreatment using P. floridanus LIPIMC996 a) without addition of

cations (control), b) the addition of CuSO4 (Cu2+

), c) the addition of

MnSO4 (Mn2+

). Hydrolysis was used the enzyme cellulase (60 FPU / g

substrate) and -glucosidase (64 pNGU/g substrate), temperature 50 C,

for 72 hours.

1.10. Oil Palm Empty Fruit Bunch Structural Changes after Pretreatment using Pleurotus floridanus and Phosphoric Acid

1.10.1. Effect of Pretreatment on Biomass Components Results of analysis of the composition and content OPEFB before and after

pretreatment combination with P. floridanus and phosphoric acid are presented Figure

7. The percentage content of components of lignocellulose OPEFB only slightly

changed due to fungal pretreatment but significantly changed due to phosphoric acid

pretreatment, and pretratment combination with fungal followed by phosphoric acid.

Hemicellulose content showed the lowest percentage in the second both pretreatment

using phosphoric acid, which is 9:09%. The reduction of total solid was showed

significant changes after pretreatment. Biological pretreatment using P. floridanus

showed the lowest dry weight (1.31%) and the lowest decrease in total carbohydrate

(7.88%) compared with the two other pretreatment.

0

10

20

30

40

50

60

70

0 7 14 21 28 35 42 49

Dig

esti

bil

ita

s (%

)

Waktu inkubasi (hari)

Kontrol Cu Mn

15

Figure 7. Profile of components of oil palm empty fruit bunches (OPEFB) after

pretreatment. ASL: acid soluble lignin, AIL: acid insoluble lignin.

The content of hemicellulose was the most affected by phosphoric acid

pretreatment and pretreatment combinations at 18%. Degradation of total solids after

phosphoric acid pretreatment was approximately 55%, whereas for the combination

of fungal pretreatment sebesaar 64% phosphoric acid. Loss of total carbohydrate of

the two treatments was 35% (fungal pretreatment) and 33% (fungal pretreatment

followed by phosphoric acid pretreatment). Based on these data fungal pretreatment

is more advantageous in terms of loss of carbohydrate level lower than phosphoric

acid pretreatment and fungal pretreatment followed by phosphoric acid pretreatment.

Fungal pretreatment application to OPEFB provide a greater amount of carbohydrates

and relatively more environmentally friendly than the other two pretreatment.

1.10.2. Pretreatment Effects on Structures of OPEFB The structural changes of OPEFB were analysed based on FTIR spectra of the

untreated and pretreated materials. The results are shown in Figure 8. Determination

and shifting each band corresponding to the literature listed in Table 2. Fourteen

bands were conserved in all of the samples in the range of 6001,800 cm1 and 2,8003,700 cm1. Bands at wavenumbers 2,918, 2,985, and 648 cm1 with high intensity were only found in untreated and fungal pretreated OPEFB. Bands that only

appeared in samples pretreated with phosphoric acid and fungal followed by

phosphoric acid were 1,224, 998 and 666 cm1

.

16

Figure 8. FTIR spectra of oil palm empty fruit bunches (OPEFB) in the

wavelength range from (a) 2800-3800 cm-1

and (b) 600-1800 cm-1

.

Information line: without pretreatment (red line), fungal pretreatment

(green line), phosphoric acid pretreatment (light blue line), fungal

pretreatment followed by phosphoric acid (brown line).

Tabel 2. Assignments of IR band maxima to various components of oil palm

empty fruit bunches according to literature.

Untreated

OPEFB

Fungal

pretreatment

Phosphoric

acid

pretreatment

Fungal

followed by

phosphoric

acid

pretreatment

Assignments Source Ref.

648 666 666 667

C-O out-of-

plane bending

mode

Cellulose

(Schwanni

nger et al.

2004)

716 - - -

Rocking

vibration CH2

in Cellulose I

Cellulose

(Schwanni

nger et al.

2004)

770 770 769 769 CH2 vibration

in Cellulose I Cellulose

(Schwanni

nger et al.

2004)

849 851 850 851

C-H out of

plane

deformation in

position 2,5,6

G-Lignin (Fackler et

al. 2010)

897 896 895 895

Anomere C-

groups C(1)-H

deformation,

ring valence

vibration

Polisakari

da

(Fackler et

al. 2010,

Fengel

1992)

- - 998 997 C-O valence

vibration

(Schwanni

nger et al.

2004)

17

Untreated

OPEFB

Fungal

pretreatment

Phosphoric

acid

pretreatment

Fungal

followed by

phosphoric

acid

pretreatment

Assignments Source Ref.

1,032 1,033 1,022 1,022

Aromatic C-H

in plane

deformation,

G > S; plus C-

O deformation

in primary

alcohols; plus

C=O stretch

(unconj.)

Lignin

(Schwanni

nger et al.

2004)

1,159 1,159 1,158 1,158

C-O-C

assimetric

valence

vibration

Polisakari

da

(Fackler et

al. 2010)

- - 1,224 1,223

C-C plus C-O

plus C=O

strech; G

condensed >

G etherified

Polisakari

da

(Fackler et

al. 2010,

Fengel

1992)

1,241 1,237 1,243 1,245

C=O stretch,

OH i.p.

bending

(Faix O.

and

Bttcher

1992)

1,266 1,267 1,267 1,267 G-ring plus

C=O strectch G-Lignin

(Faix O.

1991)

1,321 1,326 1,315 1,315

O-H blending

of alcohol

groups

Karbohidr

at

(Fackler et

al. 2010)

1,375 1,371 1,370 1,372

C-H

deformation

vibration

Cellulose (Fengel

1992)

1,418 1,418 1,420 1,419

Aromatic

skeletal

vibrations

with C-H in

plane

deformation

CH2

scissoring

Lignin

(Faix

Oskar et al.

1991)

1,462 1,457 1,455 1,459

C-H in pyran

ring

symmetric

scissoring; OH

plane

Cellulose (Fengel

1992)

18

Untreated

OPEFB

Fungal

pretreatment

Phosphoric

acid

pretreatment

Fungal

followed by

phosphoric

acid

pretreatment

Assignments Source Ref.

deformation

vibration

1,511 1,507 1,506 1,506

Aromatic

skeletal

vibrations;

G > S

Lignin

(Faix

Oskar et al.

1991)

1,593 1,609 1,608 1,607

Aromatic

skeletal

vibrations plus

C=O stretch;

S>G; G

condensed >

G etherified

Lignin

(Faix

Oskar et al.

1991)

1,640 1,646 1,654 1,663

C O stretch in

conjugated p-

substituted

aryl ketones

Lignin

(Faix

Oskar et al.

1991)

1,735 1,735 1,735 1,735

CO stretch

unconjugated

(xylan)

Polisakari

da

(Faix

Oskar et al.

1991)

2,850 2,850 2,850 2,850

Asymetric

CH2 valence

vibration

(Schwanni

nger et al.

2004)

2,918 2,918 2,918 2,918

Symmetric

CH2 valence

vibration

(Schwanni

nger et al.

2004)

3,338 3,345 3,346 3,351

Hydrogen

bonded O-H

valence

vibration;

O(3)H...O(3)

intermolecular

in cellulose

Cellulose

(Schwanni

nger et al.

2004)

A strong and broad absorption was observed at a wavenumber of around 3,300

cm-1

. This wavenumber was assigned to hydrogen bonded (O-H) stretching

absorption. O-H stretching region at a wavenumber of 3,0003,600 cm-1 of OPEFB spectra was more identical to the O-H stretching region from cellulose I than

cellulose II. The valence vibration of hydrogen-bonding of OH groups of cellulose I

is the sum of three different hydrogen-bonds: intramolecular hydrogen bond of 2-

OHO-6, intramolecular hydrogen bond of 3-OHO-5, intermolecular hydrogen bond of 6-OHO-3 (Schwanninger et al. 2004). Relatively high band in this

19

wavelength interval decreased as a result of a decrease in hydrogen bonding and

contains cellulose . Hydrogen bonding bands in the wavelength range of 2800-3800

cm-1

shows the same trend as the degradation of cellulose after pretreatment. The

highest cellulose loss was observed in fungal followed by phosphoric acid

pretreatment as indicated with the lowest intensity on O-H stretching absorption.

A strong intensity band at wavenumbers 2,985 and 2,918 cm-1

was found in

untreated and fungal pretreated OPEFB at these two wavenumbers are similar to IR

spectra from hardwood and hardwood lignin (Fackler et al. 2010), which suggests

that lignin structure in OPEFB is similar to hardwood lignin. Decrease in IR intensity

at both wavelengths are on OPEFB who received pretreatment with phosphoric acid

and fungal pretreatment combination with phosphoric acid showed a large change in

the structure of the CH2 groups.

Infrared spectra in the wavelength range of 1,150 and 1,750 cm-1

clearly

shows two distinct spectral groups (Figure 8b). The band at a wavenumber of around

1,735 cm-1

was assigned to an unconjugated carbonyl originated from the uronic acid

of the xylans in hemicellulose (Fackler et al. 2010). In this peak, there may exist

linkages between lignin and carbohydrate (Fengel 1992). IR intensities at this

wavenumber diminished after fungal pretreatment. Interestingly, it showed shoulder

peaks after phosphoric acid pretreatment and fungal followed by phosphoric acid

pretreatment. These peaks at wavenumber 1,735 cm-1

confirmed slight changes in

hemicellulose content after fungal pretreatment and a high loss of hemicellulose after

phosphoric acid pretreatments and fungal followed by phosphoric acid pretreatment

(Figure 8b).

Structural changes in lignin and loss of aromatic units were shown by the

intensities in the changes in the 1,646, 1,593 and 1,506 cm-1

bands. Fungal

pretreatment increased the intensity of the 1,646 cm-1

band and decreased the

intensity of the bands at 1,593 and 1,506 cm-1

. These changes suggest a split between

the benzylic - and -carbon atoms by fungal pretreatment (Fackler et al. 2010). Both phosphoric acid pretreatment and fungal followed by phosphoric acid

pretreatment showed similar intensities for the bands at 1,646, 1,607, 1,593, and

1,506 cm-1

. These spectra explained the fact shown in Tables 1 and 2 that pretreated

OPEFB by phosphoric acid pretreatment and fungal followed by phosphoric acid

pretreatment had a similar loss and the percentage of ASL.

IR intensity decreased at wave numbers 1,462 and 1,418 cm-1

, but increased at

wavenumber 1,321 cm-1

after fungal pretreatment. IR intensities of these bands were

reduced after phosphoric acid pretreatment and fungal followed by phosphoric acid

pretreatment. Different intensities were also found in the bands near wavenumbers

1,267 and 1,236 cm-1

. The intensities at these bands did not change after fungal

pretreatment, but reduced after phosphoric acid pretreatment. A band at 1,267 cm-1

was assigned to the guaiacyl of lignin. A band at 1,235 cm-1

was attributed to a

combination of a deformation of syringyl and cellulose. The decrease in intensity at

wavenumber 1,235 cm-1

was greater than that at wavenumber 1,267 cm-1

after

20

phosphoric acid pretreatment. This suggests that syringyl was more solubilized by

phosphoric acid than guaiacyl lignin.

The band at wavenumber 1,375 cm-1

was assigned to C-H deformations in

cellulose and hemicellulose. The intensity of this band was slightly decreased after

fungal pretreatment and it showed a slight loss of cellulose and hemicellulose

content. A higher decrease in intensity was found after phosphoric acid pretreatment,

which could be related to the high loss of hemicellulose content. Decreasing

intensities were also found in the band at wavenumber 1,159 cm-1

, which was

assigned to C-O-O- > C-O-C asymmetric vibration of cellulose and hemicellulose.

All the pretreated OPEFB samples showed lower intensities than the untreated

OPEFB. Changes in intensity were also found in the band at around wavenumber

1,032 cm-1

that was assigned to the C-O stretch in cellulose and hemicellulose.

Intensity of this band was slightly increased after fungal pretreatment. On the other

hand, it shifted to 1,021 cm-1

and decreased in intensity after phosphoric acid

pretreatment. The shifting and decreasing at this band might be attributed to

decreased hemicellulose content after phosphoric acid pretreatment.

Figure 9. FTIR spectra (a) and second derivative spectra (b) at wavenumber 770

cm1

(CH2 vibration in Cellulose I) and 716 cm1

(CH2 vibration in

Cellulose I ). Lines assignment were: untreated (red line), fungal

pretreatment (green line), phosphoric acid pretreatment (light blue line),

fungal followed phosphoric acid pretreatment (light brown line).

The peak at a wavenumber around 895 cm-1

was assigned to C-H-O stretching

of the -(1-4)-glycosidic linkage. Intensities of this peak were increased after fungal

pretreatment and phosphoric acid pretreatment, but decreased by fungal followed by

phosphoric acid pretreatment (Figure 8b) shows peaks at wavenumbers of around 750

cm-1

and 716 cm-1

that were assigned to rocking vibration CH2 in cellulose I and

cellulose I , respectively. Crystalline cellulose I is composed of two allomorphs,

Cellulose I (triclinic) and Cellulose I (monoclinic) (O'Sullivan 1997). Peaks at 769

cm1

were clearly observed in all spectra. A clear peak at wavenumber 716 cm-1

was

21

only found in untreated OPEFB spectra which then became a shoulder peak after

pretreatments. Second derivative spectra revealed that peaks at a wavenumber around

769 cm-1

for cellulose I was showed constant intensities after pretreatment.

However, peaks at a wavenumber of 716 cm-1

for cellulose I were decreased

significantly after pretreatment ( Figure 9b ).

Different methods have been proposed to characterize and quantify the

crystallinity of cellulose using the ratio of the intensities of certain bands at the IR

spectra, i.e,: 2,900, 1,429, 1,372, 894 and 670 cm-1

. The IR A1418/A895 known as

Lateral Order Index (LOI) is the ratio between absorbance at wavenumber 1,418 and

895 cm-1

(Hurtubise and Krassig 1960, O'Connor et al. 1958). LOI value of untreated,

fungal pretreated, phosphoric acid pretreated, and fungal followed by phosphoric acid

pretreated OPEFB are 2.78, 1.42, 0.67, and 0.60, respectively. Untreated OPEFB has

the highest value and the greatest decrease was achieved by phosphoric acid

pretreatment. There is no significant difference between the LOI values of phosphoric

acid and fungal pretreatment followed by phosphoric acid pretreatment. The LOI

showed a linear correlation with the hemicellulose content The correlation of LOI and

hemicelluloses was probably due to the fact that the band at 894 cm-1

was assigned to

the anomeric carbon group frequency in hemicellulose and cellulose (O'Connor et al.

1958). Results of this analysis also was suggests that the crystallinity of cellulose

associated with hemicellulose content.

1.10.3. Effect of pretreatment on OPEFB Morphology Photomicrographs of untreated OPEFB and fungal-pretreated OPEFB are

presented in Figure 10. The strand surface of untreated OPEFB has round-shaped

spiky silica-bodies. The silica bodies were found in great number and attached

relative uniformly around the fibre surface. Fungal pretreated OPEFB shows that

some of silica bodies were removed from the strand surface and left empty holes at

the bottom of silica-bodies creatures (Figure 10b). The surfaces of fungal pretreated

OPEFB are rugged and partially broken faced. Mycelium growth was found in fungal

pretreated OPEFB (Figure 10c,d). Mycelium grows outside and penetrates inside the

OPEFB strand.

Figure 11 presents photomicrographs of untreated, fungal pretreated,

phosphoric acid pretreated, and fungal followed by phosphoric acid pretreated

OPEFB after being ball-milled. The particle size of pretreated OPEFB varied.

Untreated and fungal-pretreated OPEFB showed larger particle size compared to

OPEFB pretreated by phosphoric acid and fungal followed by phosphoric acid

pretreatment (Figure 11a,b).

22

Figure 10. Fiber surface of untreated Oil palm empty fruit bunches (OPEFB). (a)

untreated OPEFB, (b) fungal pretreated OPEFB, (c) fungal pretreated

OPEFB strand covered by fungal mycellium, (d) cross section of fungal

pretreated OPEFB. SB = silica body, EH = empty hole, M = mycelium.

Some silica bodies were partially removed in the untreated OPEFB, but the

removal was higher in the fungal pretreated OPEFB. It seems that silica bodies were

easier to remove by ball mill in the fungal pretreated OPEFB than in the untreated

OPEFB. Biological pretreatment was likely to loosen the bond between silica bodies

and the surface of OPEFB fibers. Silica bodies were not found on both of the OPEFB

preetreated using phosphoric acid. Phosphoric acid and fungal followed by

phosphoric acid pretreated samples shows small size and non-uniform particles

(Figure 11 c, d). Strands of OPEFB are completely broken after phosphoric acid

pretreatment and fungal followed by phosphoric acid pretreatment. The

photomicrographs revealed that strands of OPEFB pretreated by phosphoric acid and

fungal followed by phosphoric acid pretreatment were weaker and easier to grind

than OPEFB strands pretreated by the other methods.

EM

SB a b

M c

M

d

23

Figure 11. Morphological changes of OPEFB surface before and after pretreatment.

All samples were size-reduced using ball milling before hydrolysis and

fermentation. (a) untreated OPEFB, (b) fungal pretreated OPEFB, (c)

phosphoric acid pretreated OPEFB, (d) fungal followed by phosphoric

acid pretreatmented OPEFB.

1.10.4. Cellulose Digestibility Figure 12 shows the digestibility of untreated and pretreated OPEFB after 72

h enzymatic hydrolysis. The digestibility was calculated based on initial cellulose

content prior to hydrolysis. Digestibilitas is calculated based on the initial cellulose

content OPEFB before pretreatment (equation 2). It is shown that untreated OPEFB

had very low digestibility (4.66%), which could be caused by its high lignin and high

hemicellulose contents, as well as high crystallinity of cellulose.

Digestibilitas OPEFB example that gets pretreatment is as follows: 18.85%

(fungal pretreatment), 29.15% (phosphoric acid pretreatment), and 34.64%

(pretreatment mushroom-phosphoric acid). Digestibilitas it increased respectively by

400% (pretreatment mushrooms), 630% (phosphoric acid pretreatment), and 740%

(pretreatment mushroom-phosphoric acid) times compared with digestiblitas OPEFB

who did not receive pretreatment. Moreover, it is comparable to digestilitas

digestilitas OPEFB after a pretreatment with ammonia (Ammonia Fiber Expansion,

AFEX) pretreatment (58%) (Lau et al. 2010), alkali pretreatment (69.69%)

(Piarpuzn et al. 2011), pretreatment superheated steam (66.33%) (Bahrin et al. 2012)

and sodium hydroxide-sodium pretreatment hypoclorite (60%) (Hamzah et al. 2011).

Digestibilitas OPEFB after biological pretreatment for 28 days with P. floridanus

Digestibility higher than that in the Japanese pine-pretreatment with Stereum

a b

c d

24

hirsutum for eight weeks (13.56%) (Lee et al. 2007).

Figure 12. Digestibility of cellulose (%) of oil palm empty fruit bunches (OPEFB)

in the enzymatic hydrolysis process (based on initial cellulose content

after pretreatments). Error bars are standard deviation. Hidrolysis was

used commercial enzyme Cellic CTec2, at 50oC, for 72 h.

The digestibility of OPEFB after pretreatment has an inverse correlation with

LOI. Digestibility is enhanced as crystallinity of the cellulose is reduced as shown by

the lower LOI value. The IR spectra of fungal pretreatment samples indicate that the

fungus might attack the linkages between lignin and carbohydrate that exist in

hemicellulose.

Results of correlation analysis between digestibilitas with LOI values indicate

an inverse correlation where digestibilitas OPEFB increases with decreasing value of

LOI. Increasing cellulose digestibility was due to several changes in the structure of

OPEFB, such as the decreasing of hemicellulose content, breaking down the bonds

between lignin and cellulose, decreasing of cellulose crystallinity and increasing of

cellulose I. Cellulose I is meta-stable and more reactive than cellulose I (O'Sullivan 1997). This possibility makes OPEFB more reactive and more easily

hydrolyzed.

OPEFB pretreated by phosphoric acid and fungal followed by phosphoric acid

methods showed relatively high lignin proportion up to 44.66%. This finding stresses

the fact that lignin seems not the only recalcitrant factor of OPEFB. Available surface

area and accessibility to cellulose of OPEFB after pretreatment contribute to

improved digestibility of lignocellulosic materials (Rollin et al. 2010).

1.10.5. Bioethanol Production Production of bioethanol (ethanol yield) was shown in Figure 13. Bioethanol

production by SSF method of sample OPEFB showed a similar pattern with OPEFB

digestibility (Figure 12). Production of bioethanol from the highest were combination

fungal pretreatment-phosphoric acid, phosphoric acid pretreatment, fungal

4,66

18,85

29,15

34,64

05

1015202530

3540

Kontrol Pretreatment

Jamur

Pretreatment

asam fosfat

Pretreatment

jamur-asam

fosfat

Dig

esti

bil

ita

s se

lulo

sa (

%)

25

pretreatment, and without pretreatment.

Figure 13. Percentage of bioethanol production from the theoretical maximum

production (ethanol yield ) of oil palm empty fruit bunches (OPEFB) by

the method of SSF (simultaneous saccharification and fermentation).

Yeast will ferment glucose resulted from enzymatic hydrolysis of cellulose to

ethanol. Cellulose digestibility cellulose will increase in line with increased

production of ethanol by yeast. Increase the ethanol yield of each treatment at the 72h

than control treatment was 222 % (fungal pretreatment), 642 % (phosphoric acid

pretreatment), and 701 % (fungal pretreatment and phosphoric acid). Ethanol yield

has significant positive linear correlation ( r2 = 0.99 ) with OPEFB digestibility which

means that the increasing digestibility will be followed by an increasing in ethanol

production.

Ethanol yield resulting from this study is higher than the yield of ethanol that

reported in some literature. Yield of ethanol from biological pretreated OPEFB were

6 g / L higher than the yield of ethanol from the alkali pretreated OPEFB ( 4 g / L )

(Piarpuzn et al. 2011). Increasing the yield of ethanol of the combination pretreated

OPEFB increased 7.01 times. This increasing was higher than the increasing of water

hyacinth (Eichhornia crassipes) were pretreated with alkali and WRP which the

increasing was only 1.34 times (Ma et al. 2010).

CONCLUDING REMARKS

Third WRP isolates have varying selectivity to degrade lignin, cellulose and

hemicellulose. Pleurotus floridanus isolates showed the highest degradation of lignin

and lowest cellulose degradation. P. floridanus was more selectively to degrade lignin

than other isolates. The addition of Cu2+

and Mn2+

could increase lignin degradation

by P. floridanus. Lignin content was degraded up to 46.62 % within 42 days of

incubation. Physical, chemical, and structural of OPEFB was changing after

pretreated with P. floridanus, phosphoric acid , and a combination of biological and

0

10

20

30

40

50

60

70

80

90

0 24 48 72 96

Yie

ld E

tan

ol

(%)

Jam ke-Kontrol Pretreatment Jamur

Pretreatment Jamur - Asam Fosfat Pretreatment Asam Fosfat

26

phosphoric acid pretreatment. Some functional groups mainly syringyl and guaiacyl lignin units undergo significant changes. Cellulose crystallinity of OPEFB was decreased . Important structural changes observed by FTIR analysis were reduction of hydrogen bond (OH), unconjugated carbonyl absorption, the absorption peak ( peak ) for cellulose and hemicellulose , and cellulose peaks decrease I. Digestibility OPEFB and ethanol production has increased very significantly on a combination of biological pretreatment and phosphoric acid. Degradation of lignin and hemicellulose, reduction of cellulose crystallinity, decreased cellulose I, and particle size reduction and contribute to the increase in ethanol production digestibilitas.

Refernces Alvira P, Toms-Pej E, Ballesteros M, Negro MJ. 2010. Pretreatment technologies

for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresource Technology 101: 4851-4861.

Anderson WF, Akin DE. 2008. Structural and chemical properties of grass lignocelluloses related to conversion for biofuels. J Ind Microbiol Biotechnol 35: 355366.

Bahrin EK, Baharuddin AS, Ibrahim MF, Razak MHA, Sulaiman A, Abd-Aziz S, Hassan MA, Shirai Y, Nishida H. 2012. Physicochemical property changed and enzymatic hydrolysis enhancement of oil palm emtpy fruit bunches treated with superheated steam. BioResources 7(2): 1784-1801.

Bajpai P. 2004. Biological Bleaching of Chemical Pulps. Critical Reviews in Biotechnology 24 (1): 1-58.

Camarero S, Bockle B, Martnez M, Martnez A. 1996. Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius. Applied and Environmental Microbiology 62: 1070-1072.

Chen M, Yao S, Zhang H, Liang X. 2010. Purification and Characterization of a Versatile Peroxidase from Edible Mushroom Pleurotus eryngii. Chinese Journal of Chemical Engineering 18: 824-829.

Datta R. 1981. Acidogenic Fermentation of Lignocellulose-Acid Yield and Conversion of Components. Biotechnology and Bioengineering XXIII: 2167-2170.

de Jong E, Chandra RP, Saddler JN. 1997. Effect of a Fungal Treatment of The Brightness and Strength Properties of A Mechanical Pulp from Douglas-Fir. Bioresource Technology 61: 61-68.

Dowe N, McMillan JD. 2008. SSF Experimental Protocols Lignocellulosic Biomass Hydrolysis and Fermentation. National Renewable Energy Laboratory. Report no. NREL/TP-510-42630.

Fackler K, Stevanic JS, Ters T, Hinterstoisser B, Schwanninger M, Salmn L. 2010. Localisation and characterisation of incipient brown-rot decay within spruce

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wood cell walls using FT-IR imaging microscopy. Enzyme and Microbial

Technology 47: 257-267.

Faix O. 1991. Classification of Lignins from Different Botanical Origins by FT-IR

Spectroscopy. Holzforchung 45: suppl. 21-27.

Faix O, Bttcher JH. 1992. The influence of particle size and concentration in

transmission and diffuse reflectance spectroscopy of wood. Holz als Roh- und

Werkstoff 50: 221-226.

Faix O, Bremer J, Schmidt O, Tatjana SJ. 1991. Monitoring of chemical changes in

white-rot degraded beech wood by pyrolysisgas chromatography and Fourier-transform infrared spectroscopy. J Anal Appl Pyrolysis 21: 147-162.

FAOSTAT. 2012. Food and Agriculture Organization of the United Nations.

(Accessed 28 October 2012

http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor)

Fengel D. 1992. Characterization of Cellulose by Deconvoluting the OH Valency

Range in FTIR Spectra. Holzforschung 46: 283-285.

Goudopoulou A, Krimitzas A, Typas MA. 2010. Differential gene expression of

ligninolytic enzymes in Pleurotus ostreatus grown on olive oil mill

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TITLE PAGEABSTRACTINTRODUCTIONMATERIALS AND METHODS1.1. Microorganisms and Medium1.1.1. Microorganisms1.1.2. Media1.1.3. Tandan Kosong Kelapa Sawit (TKKS)

1.2. Phases of Research1.3. Pretreatment Methods1.4. Phosphoric Acid Pretreatment Methods1.5. Enzymatic Hydrolysis1.6. Simultaneous Saccharification and Fermentation1.7. Analytical Methods

RESULTS AND DISCUSSIONS1.8. White-rot fungi Selection for Biological Pretreatment of Oil Palm Empty Fruit Bunch1.9. Effect of Addition of Manganese (Mn2+) and copper (Cu2+) on Biological Pretreatment of Oil Palm Empty Fruit Bunch Using Pleurotus floridanus LIPIMC9661.9.1. Effect of Biological Pretreatment on Dry Weight and Lignocellulosic Components1.9.2. Effect of Biological Pretreatment on Physical and Structural Characteristics1.9.3. Effect of Biological Pretreatment on Digestibility

1.10. Oil Palm Empty Fruit Bunch Structural Changes after Pretreatment using Pleurotus floridanus and Phosphoric Acid1.10.1. Effect of Pretreatment on Biomass Components1.10.2. Pretreatment Effects on Structures of OPEFB1.10.3. Effect of pretreatment on OPEFB Morphology1.10.4. Cellulose Digestibility1.10.5. Bioethanol Production

CONCLUDING REMARKSReferences